Quorum sensing (QS) has evolved as a means for bacterial communities to regulate gene expression in response to environmental cues in a coordinated manner. Each cell of the population produces and secretes signaling molecules, called autoinducers, and responds to these molecules, which thus serve as indicators of the population density. QS circuits control complex bacterial behaviors, such as bioluminescence, virulence, antibiotic resistance, and biofilm formation. QS systems have been discovered in Gram-negative and -positive bacteria and a variety of molecules, e.g. oligopeptides and acyl homoserine lactones, have been identified as autoinducers. A distinct third class of QS molecules are autoinducers derived from the precursor (S)-4,5-dihydroxy-2,3-pentanedione (DPD). So far, two distinct members of this family have been structurally identified. The luxS gene encoding the enzyme responsible for the final step in the biosynthesis of DPD, has been identified in over 55 bacterial species, both Gram-positive and -negative bacteria, including many clinically relevant pathogens. We have designed a series of chemical, biochemical, and biological experiments to examine and evaluate the molecular mechanisms that define AI-2 recognition by bacterial cells. In addition these investigations will help elucidate the bioactivity of DPD in bacterial systems, as well as to obtain molecules that possess agonistic or antagonistic signaling activity. The specific aims of our proposal are: 1) Synthesis of DPD/AI-2 Agonists and Antagonists; 2) Mechanistic Investigations of DPD-mediated Effects in Pathogenic Bacteria; 3) Elucidation of the Cellular Recognition of AI-2; and 4) Proteomic analysis of AI-2 Quorum Sensing-regulated Processes. In total, we believe that the use of synthetic DPD and systematically designed analogs of DPD/AI-2 in the experimental methodologies outlined in this proposal will provide new biochemical and microbiological insights into DPD-/AI-2-based QS systems and will help to evaluate the therapeutic value of AI-2-dependent QS as new leads for the antibacterial strategies. PUBLIC HEALTH RELEVANCE: Microbial cell-to-cell signaling has been coined quorum sensing. It controls many bacterial processes, such as antibiotic resistance and biofilm formation, which represents a majority of disease states where chronic bacterial infection leads to tissue destruction and loss of organ function. This includes diseases as diverse as dental caries, wound infections, and even anthrax. This proposal is designed to gain insight into the molecular details of quorum sensing and to develop countermeasures to prevent quorum sensing signaling in a wide number of bacteria.